The human carcinoembryonic antigen (CEA), which is expressed in colorectal, pancreatic, gastric, breast, and non-small cell lung cancers is a potential target for specific immunotherapy using recombinant vaccines. Previous studies have shown that when the CEA gene is placed into vaccinia virus, the recombinant vaccine, designated rV-CEA, can elicit T-cell responses both in rodents and non-human primates. Due to the uniqueness of human HLA motifs, these previous studies could not predict if rV-CEA could elicit CEA specific T-cell responses in humans. Peripheral blood lymphocytes (PBLs) obtained from patients with metastatic carcinoma, both pre- and post- vaccination with rV-CEA, were analyzed for T-cell response to specific 9-11 mer CEA peptides selected to conform to human HLA class I-A2 motifs. While little or no T-cell growth was seen from pre- immunization PBLs of patients pulsed with CEA peptides and IL-2, T- cell lines were obtained from PBLs of patients post-vaccination, after 1-3 cycles of stimulation. Cytolytic T-cell lines from several A2 patients (post-vaccination) were established with one peptide (designated CAP-1) and the T-cell line (designated V24T) from one patient was chosen for detailed analysis. V24T was shown to be a CD8+/CD4+ double positive and to synthesize TNF-` (>300pg/ml) when exposed to the 9 amino acid CAP-1 peptide. When autologous EBV- transformed B-cells were either incubated with CAP-1 peptide, or transduced with the CEA gene using a retroviral vector, they were lysed by the V24T cell line. Allogeneic non-A2 EBV transformed B- cells were not lysed by V24T when incubated with CAP-1. A human colon carcinoma cell line which is CEA positive and A2 positive, was also lysed by the V24T cell line while two non-A2 CEA positive colon carcinoma cell lines were not. To further define the class I HLA-A2 restricted nature of the V24T cytotoxicity, a non-A2 CEA expressing colon carcinoma cell line was infected with a recombinant vaccinia virus expressing the HLA class I-A2 gene and was shown to become susceptible to V24T lysis. Cells infected with vector alone were not lysed. These studies demonstrate for the first time, (a) the ability to generate a human cytolytic T-cell response to specific epitopes of CEA, (b) the class I HLA-A2 restricted nature of the T-cell mediated lysis, and (c) the ability of human tumor cells to endogenously synthesize CEA so as to present a specific CEA peptide in the context of MHC for T-cell lysis. These findings have implications in the development of specific second generation cancer immunotherapy protocols. We also investigated whether human T lymphocytes are able to distinguish the determinants created by point-mutated p21 ras proteins from the normal ras. Cellular immunity to 3 synthetic 13-mer peptides representing amino acids 5-17 of mutated p21 ras proteins with an exchange of normal glycine (G12) at position 12 by valine (V12), cysteine (C12) or aspartic acid (D12) was studied. Four human TCLs from different individuals were established by in vitro peptide stimulation. All TCLs were CD3+, CD4+ and CD8- phenotype. Specificity of the TCLs to stimulating peptide was observed as a proliferative response using the corresponding peptides but not peptides reflecting other mutations or normal ras (G12). Human TCLs all responded to corresponding peptides with the production of IFN-g and IL-6. Using V12 specific TCLs, EBV transformed autologous B cell lines could be lysed when incubated with V12 peptide. Furthermore, when the autologous EBV transformed B cell line was transduced with the p21 ras oncogene containing the V12 mutation in a retroviral vector, these cells became susceptible to lysis by autologous V12 specific TCL. These studies indicate that a T cell specific immune response to mutated p21 ras protein could be elicited from PBLs, suggesting point mutated ras p21 as a potential target for specific immunotherapy of human cancers.